Little is known about the cis and trans elements that determine the initiation of chromosomal DNA replication in metazoans. Indeed by contrast to what is understood about metazoan gene expression cisacting signals (e.g. enhancers and promoters) the DNA elements that define origins of replication (ori's) are undetermined. A key concept emerging from the Papova virus DNA replication field is that site specific DNA binding proteins can function as molecular chaperones for initiator proteins and that these molecules can operate both as transcription and replication factors. We propose to test the hypothesis that such ancillary factors govern cellular DNA replication and are at the center of the regulation of ori usage in metazoans. In yeast cells the origins of DNA replication are determined largely by a heteromeric protein complex called ORC. We have previously cloned the genes and reconstituted from recombinant vectors a biochemically active Origin recognition complex (ORC) from drosophila. Our evidence is that pure ORC needs protein co- factors to bind to DNA site specifically and that such factors are extremely limited (or nonexistent) in the early embryo. The aims of this proposal are thus aimed at unraveling the activities of ORC and its interactions with other key initiator proteins such as Cdc6, with the central goal of understanding tissue specific origin usage in a metazoan. The specific aims of this proposal are thus to identify proteins that interact with ORC to allow for specific DNA binding and to reconstitute this activity in vitro. We shall clone the genes encoding for these proteins and endeavor to show that their binding sites are critical in vivo for site specific gene amplification and DNA replication. We propose to assay the activities of the various ORC subunits by application of our in-vitro DNA replication system utilizing extracts depleted of endogenous ORC and rescued either with wild type complex or mutant complex. This system also provides a powerful approach to study the Cdc6 protein and its interactions with ORC. We will test the notion that phosphorylation of Cdc6p and ATP binding mediate the interactions. We propose to critically test the model that ORC is multi-functional and paztidpates not only in DNA replication control but in chromosome folding and gene expression.